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United States Patent |
5,320,950
|
Grundmann
,   et al.
|
June 14, 1994
|
DNA encoding anticoagulative protein PP4-X, and its preparation and use
Abstract
The protein PP4-X, whose amino acid sequence and the DNA sequence coding
for this have been determined, has anticoagulative properties and can be
prepared by genetic manipulation. PP4-X is used for therapeutic and
diagnostic purposes.
Inventors:
|
Grundmann; Ulrich (Lahntal-Grossfelden, DE);
Abel; Karl-Josef (Marburg, DE);
Amann; Egon (Marburg, DE)
|
Assignee:
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Behringwerke Aktiengesellschaft (Marburg/Lahn, DE)
|
Appl. No.:
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985912 |
Filed:
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December 4, 1992 |
Foreign Application Priority Data
Current U.S. Class: |
435/69.1; 435/69.2; 435/320.1; 514/2; 530/350; 536/23.4; 536/23.5 |
Intern'l Class: |
C12N 015/12; C12N 015/62; C12N 001/11 |
Field of Search: |
536/23.5,24.31,23.4
435/240.2,320.1,69.1,69.7
|
References Cited
U.S. Patent Documents
4874743 | Oct., 1989 | Wallner et al. | 514/12.
|
4937324 | Apr., 1990 | Fujikawa et al. | 530/397.
|
Foreign Patent Documents |
WO86/04094 | Jul., 1986 | WO.
| |
WO88/05659 | Aug., 1988 | WO.
| |
Other References
Geisow, J. J. (1986) FEBS Lett. 203:99-103.
Creutz, C. E., et al. (1987) J. Biol. Chem. 262:1860-68.
Darnell, J. E., et al. (1986) Molecular Cell Biology, Sci. Am. Press, New
York, p. 373.
Weber et al., "The Amino Acid Sequence of Protein II and its
Phosphorylation Site for Protein Kinase C . . . " The EMBO Journal,
6:1599-1604, 1987.
Iwasaki et al., "Structure and Expression of cDNA for an Inhibitor of Blood
Coagulation . . . ", J. Biochem. 102: 1261-1273 (1987).
Crumpton et al., "Protein Terminology Tangle", Nature, 345:212 (1990).
|
Primary Examiner: Hill, Jr.; Robert J.
Assistant Examiner: Fitzgerald; David L.
Attorney, Agent or Firm: Finnegan, Henderson Farabow, Garrett & Dunner
Parent Case Text
This is a division of application Ser. No. 07/841,293, filed Feb. 28, 1992,
now U.S. Pat. No. 5,202,419, which is a continuation of application Ser.
No. 07/265,843, filed Nov. 1, 1988, now abandoned.
Claims
We claim:
1. An isolated nucleic acid molecule having a sequence which encodes the
amino acid sequence of PP4-X shown in FIG. 2.
2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule
has the sequence shown in FIG. 2.
3. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule
is a genomic clone.
4. A cultured cell transformed with the nucleic acid molecule of claim 1.
5. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule
is a cDNA.
6. A process for the preparation of PP4-X comprising inserting the cDNA of
claim 5 into an expression system and bringing about its expression
therein.
7. A vector comprising a nucleic acid molecule having a sequence which
encodes the amino acid sequence of PP4-X shown in FIG. 2.
8. DNA encoding a fusion protein comprising the PP4-X amino acid sequence
shown in FIG. 2 and a heterologous polypeptide moiety.
9. A vector comprising the DNA of claim 8.
10. A cultured cell transformed with the DNA of claim 8.
11. A process for the expression of a PP.sub.4 -X fusion protein comprising
inserting the DNA of claim 8 into an expression system and bringing about
its expression therein.
Description
DESCRIPTION
H. Bohn (German Offenlegungsschrift DE-A 3,315,000) was the first to
describe the protein PP4 from human placentae and to characterize it by
means of physicochemical parameters. Various oligonucleotides coding for
PP4 peptides have now been used to search for the cDNA for PP4 in a
placental cDNA bank. Surprisingly, a cDNA which codes for a protein which
differs from PP4 has been found. Hence the protein resulting from the cDNA
described herein is called PP4-X. The amino acid sequence, as well as the
protein structure with 4 repeats, resembles that of the proteins
lipocortin I and II.
The proteins lipocortin I and II or calpactin II and I have recently been
disclosed as inhibitors of phospholipase A 2 (F. Davidson et al. (1987),
J. Biol. Chem., 1698-1705; for review see: M. J. Geisow and J. H. Walker
(1986), Trends in Biological Sciences 11, 420-423). Whereas lipocortin I
acts as a substrate for EGF receptor tyrosine-protein kinase, lipocortin
II is the human protein analogous to pp36, isolated from embryonal chicken
fibroblasts or bovine intestinal epithelial cells, as principal substrate
of pp60.sup.src kinase (K.-S. Huang et al. (1986) Cell 46, 191-199).
Lipocortin II is, together with actin, a constituent of the internal
membrane framework and external cytoskeleton. Lipocortin I has potent
antiinflammatory properties, analogous to the corticosteroids, and it is
presumed that the synthesis of these proteins is induced by
corticosteroids (G. Cirino et al. (1987) Nature 328, 270-272). DNA
sequence analyses and partial amino acid sequence analyses of the two
proteins have led to both the nucleic acid sequences and the derived
protein sequences being known (B. Wallner et al. (1986) Nature 320, 77-81
and K.-S. Huang et al., loc. cit.). The two sequences resemble one another
greatly and, moreover, each has four repeat structures which contain a
consensus sequence of about 20 amino acids. These four repeats in a single
basic amino acid sequence are probably necessary for the membrane-binding
properties--in the presence of Ca.sup.2+ --of the lipocortins.
Although we found PP4-X to have oligonucleotide sequences derived from PP4,
it is not identical to PP4. This is based on the fact that just one
oligonucleotide used for the search contains a base sequence which is
relatively strongly conserved both in the repeats of lipocortin I and II
and in those of PP4 and PP4-X.
PP4-X is an endogenous anticoagulant which occurs particularly in highly
vascularized tissue such as the placenta, and in vessels. The
anticoagulative properties make PP4-X suitable for use as an inhibitor in
the coagulation cascade, since it reversibly inhibits blood coagulation at
the thromboplastin stage. PP4-X binds via Ca ions to a negatively charged
phospholipid surface, from which it can be released again with EDTA. A use
resulting from this is in the prophylaxis of thrombosis, because PP4-X
selectively, effectively and reversibly interrupts coagulation, without
coagulation factors or inhibitors being respectively inactivated or
activated by proteolysis. The coagulation potential is thus retained in
full.
To obtain partial amino acid sequences and suitable oligonucleotide probes
derived therefrom, the protein PP4 was broken down into cyanogen bromide
fragments, two of which fragments (41 and 44 amino acids respectively)
were sequenced. The following sequences were found:
PP4 oligopeptide A
MKGLGTDEES ILTLLTSRSN AQRQEISAAF KTLFGRDLLD D
PP4 oligopeptide B
MLVVLLQANRD PDAGIDEAQV EQDAQALFQA GELKXGTDEE KFI
On the basis of statistical data of R. Lathe (J. Mol. Biol. (1985) 183,
1-12), from the oligopeptide A was selected an oligonucleotide sequence
having 35 bases.
(PP4 oligonucleotide 125)
ATGAAGGGCC TGGGCACAGA TGAGGAGAGC ATCCT
and from oligopeptide B a sequence having 36 bases
GATGCCCAGG CCCTGTTCCA GGCTGGCGAG CTGAAG
(PP4 oligonucleotide 104).
These oligonucleotide probes were used to screen a cDNA bank which had been
prepared from mRNA from mature human placenta. The mRNA was first isolated
from the placenta, and the cDNA was prepared therefrom. The latter was
provided with EcoRI ends and ligated into the EcoRI cleavage site of the
phage vector .lambda.gt10. 46 clones which had been identified using the
abovementioned probes were analyzed further. Sequencing by methods known
per se revealed the DNA sequence which codes for PP4-X.
FIG. 1 shows the restriction map of the cDNA sequence which encodes PP4-X.
"N" designates the N terminus, "C" designates the C terminus of the coding
region, and "A (25)" designates the poly-(A) sequence of 25 bases. The
cDNA sequence represents the complete coding sequence of PP4-X.
FIGS. 2A and 2B together show the DNA sequence (coding strand) which was
found, and the amino acid sequence derived therefrom, of one clone
(PP4-X/23). The complete cDNA has a length of 1326 base-pairs and an open
reading frame of 972 base-pairs. The positions of the oligonucleotide
probes 104 and 125 which hybridize between positions 599 and 634, and 851
and 885, respectively, are marked in FIG. 2.
It is possible according to the invention for the coding cDNA to be used,
with the aid of suitable expression systems, to express PP4-X.
Furthermore, the type of modification of PP4-X can be influenced by the
choice of the host. Thus, no glycosylation takes place in bacteria,
whereas that taking place in yeast cells differs from that in higher
eukaryotic cells.
Knowing the amino acid sequence of PP4-X, it is possible to prepare, by
conventional or genetic manipulation methods, amino acid part-sequences
which can be used as antigens for the preparation of polyclonal or
monoclonal antibodies. Such antibodies can be used not only for diagnostic
purposes but also for the preparation of antibody columns with which it is
possible to separate PP4-X from solutions which contain it together with
other proteins.
It is also possible using the cDNA, or parts thereof, to isolate in a
straightforward manner from a genomic bank the genomic clone which codes
for PP4-X and which not only facilitates the expression in eukaryotic
cells but also allows further diagnostic conclusions to be drawn.
The invention is further defined in the patent claims and is explained in
detail in the examples which follow.
The following abbreviations are used, apart from those explained in the
text:
EDTA=sodium ethylenediaminetetraacetate
SDS=sodium dodecyl sulfate
DTT=dithiothreitol
BSA=bovine serum albumin
EXAMPLES
1. Isolation of RNA from human placenta
RNA was obtained from mature human placenta (method of Chirgwin et al.,
Biochemistry 18 (1979) 5294-5299). About 10 g of placental tissue were
ground in liquid nitrogen in a mortar, suspended in 80 ml of 4M
guanidinium thiocyanate containing 0.1M mercaptoethanol, and treated in a
homogenizer (Ultraturrax) at 20,000 rpm for 90 sec. The lysate was
centrifuged (Sorvall GSA rotor) at 7,000 rpm for 15 min, and the
supernatant was precipitated with 2 ml of 1M acetic acid and 60 ml of abs.
ethanol at -20.degree. C. overnight. The nucleic acids were sedimented at
6,000 rpm and -10.degree. C. for 10 min and then completely dissolved in
40 ml of 7.5M guanidinium hydrochloride (pH 7.0) and precipitated with a
mixture of 1 ml of 1M acetic acid and 20 ml of abs. ethanol. To remove the
DNA, the precipitation was repeated once more with each of the volumes
being halved. The RNA was dissolved in 12 ml of H.sub.2 O, precipitated
with a mixture of 1.2 ml of 4M potassium acetate and 24 ml of abs.
ethanol, sedimented and, finally, again taken up in 10 ml of H.sub.2 O (1
ml per g of tissue).
2. Obtaining Poly(A)-Containing Placental mRNA
To obtain poly(A)-containing mRNA, the placental RNA was fractionated by
oligo(dT)-cellulose chromatography (Aviv and Leder, Proc. Natl. Acad. Sci.
USA 69 (1973) 1408-1412) in 2 ml Pasteur pipettes in LiCl. About 5 mg of
placental RNA in buffer 1 (500 mM LiCl, 20 mM Tris (pH 7.5), 1 mM EDTA,
0.1% SDS) were applied to the column. Whereas the poly(A).sup.+ RNA was
bound to oligo(dT)-cellulose, it was possible to elute the poly(A).sup.-
RNA again. After a washing step with buffer 2 (100 mM LiCl, 29 mM Tris (pH
7.5), 1 mM EDTA, 0.1% SDS), the poly(A).sup.+ RNA (placental mRNA) was
eluted from the column with buffer 3 (5 mM Tris (pH 7.5), 1 mM EDTA, 0.05%
SDS).
For further purification, the poly(A).sup.+ RNA was adjusted to buffer 1
and again chromatographed on oligo(dT)-cellulose. The yield of placental
poly(A).sup.+ RNA after this second purification step was about 4% of the
RNA used.
3. Synthesis of cDNA From Human Placenta (Placental cDNA) and
Double-Stranded cDNA (dsDNA)
The integrity of the poly(A)-containing placental mRNA was checked in a
1.5% agarose gel before the cDNA synthesis.
Then 4 .mu.g of placental mRNA were dissolved in 65.5 .mu.l of H.sub.2 O,
denatured at 70.degree. C. for 10 min and cooled again in ice. The cDNA
was synthesized in a 100 .mu.l mixture after addition of 20 .mu.l of
RT.sub.1 buffer (250 mM Tris (pH 8.2) at 42.degree. C., 250 mM KCl, 30 mM
MgCl.sub.2), 2.5 .mu.l of 20 mM dNTP (i.e. all four deoxynucleoside
triphosphates), 1 .mu.l of oligo(dT) of 1 .mu.g/ml, 1 .mu.l of 1M DTT, 2
.mu.l of RNAs in and 8 .mu.l of reverse transcriptase (24 U/.mu.l) at
42.degree. C. for 90 min.
Double-stranded cDNA (dsDNA) was synthesized by the method of Gubler and
Hoffmann (Gene 25 (1983) 263-269). The synthesis was carried out
immediately after the cDNA synthesis by addition of 305.5 .mu.l of H.sub.2
O, 80 .mu.l of RT.sub.2 buffer (100 mM Tris (pH 7.5), 25 mM MgCl.sub.2,
500 mM KCl, 50 mM DTT, 250 .mu.g/ml BSA), 2 .mu.l of RNase H (2 U/.mu.l),
2.5 .mu.l of E. coli DNA ligase (5 U/.mu.l), 5 .mu.l of 15 mM .beta.-NAD,
and 5 .mu.l of DNA polymerase I (5 U/.mu.l) and incubation at 15.degree.
C. for 5 h. The reaction was stopped by heat-inactivation (70.degree. C.,
30 min).
After addition of 55 .mu.l of 250 .mu.M dNTP, 55 .mu.of 10 mM Tris (pH
7.5), 10 mM MgCl.sub.2, 10 .mu.g/ml BSA, 3 .mu.l of T4 DNA polymerase I (1
U/.mu.l), 2 .mu.l of RNase H (2 U/.mu.l) and 2 .mu.l of RNase A (2
.mu.g/ml) to the reaction mixture it was incubated at 37.degree. C. for a
further 13 min in order to ensure that the synthesis on the second DNA
strand was complete ("repair reaction").
4. Ligation of EcoRI Linkers to the dsDNA, and Opening of the Linkers
To set up a placental cDNA bank, the dsDNA was provided with EcoRI ends in
order to be able to ligate it into the EcoRI cleavage site of the phage
vector .lambda.gt10 (T. Maniatis et al. (1982), Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor). For this purpose, the DNA was
a) treated with EcoRI methylase in order to protect internal EcoRI cleavage
sites of the dsDNA, and
b) provided with EcoRI linkers which
c) were then opened with EcoRI.
Re a):
The methylase reaction of dsDNA was carried out directly following the
repair reaction after addition of 25 .mu.l of 500 mM EDTA (pH 8.0), 60
.mu.l of methylase buffer (100 mM NaOAc (pH 5.2), 2 mg of
S-adenosyl-L-methionine) and 2 .mu.l of EcoRI methylase (20 U/.mu.l) by
incubation at 37.degree. C. for 30 min.
The reaction mixture was extracted with phenol, and the dsDNA was
precipitated with 60 .mu.l of 4M NaOAc and 1300 .mu.l of ethanol. The
dsDNA was washed twice with 70% ethanol, extracted by shaking once with
ether, and dried.
Re b):
The EcoRI-methylated dsDNA was dissolved in 88 .mu.l of H.sub.2 O and,
after addition of 10 .mu.l of ligase buffer (500 mM Tris (pH 7.4), 100 mM
MgCl.sub.2, 100 mM DTT, 100 mM spermidine, 10 mM ATP, 1 mg/ml BSA) and 1
.mu.l of T4 DNA ligase (10 U/.mu.l), was ligated with 1 .mu.l of EcoRI
linkers (0.5 .mu.g/.mu.l) (pGG-AATTCC and pAGAATTCT) at 15.degree. C.
overnight.
Re c):
The volume of the ligase mixture was made up to 120 .mu.l with 6 .mu.l of
H.sub.2 O, 12 .mu.l of 10.times.EcoRI buffer and 2 .mu.l of EcoRI (120
U/.mu.l). The EcoRI digestion was carried out at 37.degree. C. for 2 h.
5. Removal of Unbound Linkers on a Potassium Acetate Gradient, and
Selection of the dsDNA for Size
All unbound EcoRI linkers were removed from the dsDNA by applying the EcoRI
reaction mixture in toto to a potassium acetate gradient (5-20% KOAc, 1 mM
EDTA, 1 .mu.l/ml ethidium bromide) and centrifuging (Beckman SW 65 rotor)
at 50,000 rpm and 20.degree. C. for 3 h.
The gradient was fractionated from below in such a way that the first five
fractions measured 500 .mu.l, and all the remainder measured 100 .mu.l.
The fractions were precipitated with 0.01 volume of acrylamide (2 mg/ml)
and 2.5 volumes of ethanol, washed once with 70% strength ethanol and
dried, and each was taken up in 5 .mu.l of H.sub.2 O.
To determine the size of the dsDNA, 1 .mu.l of each fraction was analyzed
in 1.5% agarose gel. In addition, the quantity of dsDNA was determined
using 1 .mu.l of each fraction.
Fractions containing dsDNA above 1000 bp were combined, and the sample was
concentrated until the final concentration was 27 .mu.g/ml.
6. Insertion of the dsDNA Into the Phage Vector .lambda.gt10, and in Vitro
Packaging Reaction
The dsDNA was inserted into the EcoRI cleavage site of the phage vector
.lambda.gt10 (Vector Cloning Systems, San Diego, Calif.) in a 4 .mu.l
ligase mixtures: 2 .mu.l of dsDNA, 1 .mu.l of .lambda.gt10.times.EcoRI (1
.mu.g/ml), 0.4 .mu.l of ligase buffer, 0.5 .mu.l of H.sub.2 O, 0.1 .mu.l
of T4 DNA ligase. The mixture was incubated at 15.degree. C. for 4 h.
To establish the placental cDNA bank in the phage vector .lambda.gt10, the
ligase mixture was subsequently subjected to an in vitro packaging
reaction with the .lambda.-lysogenic cell extracts E. coli NS 428 and NS
433 at room temperature for 2 h (Vector Cloning Systems, San Diego,
Calif.; Enquist and Sternberg, Methods in Enzymology 68, (1979), 281-298).
The reaction was stopped with 500 .mu.l of suspending medium (SM: 0.1M
NaCl, 8 mM MgSO.sub.4, 50 mM Tris (pH 7.5), 0.01% gelatine), and 2 drops
of chloroform.
7. Titer Determination and Analysis of the Placental cDNA Bank
The number of plaque-forming units (PFU) of the placental cDNA bank were
determined using competent cells of E. coli K 12 strain C600 HFL: it was
1.times.10.sup.6 PFU. About 80% of all the phages contained DNA inserts
larger than 1000 base-pairs.
8. Oligonucleotide Probes for Screening the Placental cDNA Bank
Two oligonucleotide probes were synthesized for the analysis of the
placental cDNA bank. Their sequences were derived from the amino acid
sequence of several cyanogen bromide fragments of the anticoagulative
protein PP4-X.
The manner of construction and the use of the two probes essentially
followed the rules of R. Lathe, loc. cit..
Both oligonucleotide sequences were labeled at the 5' end using T4
polynucleotide kinase in the presence of (.gamma.-.sup.32 P) ATP (about 1
.mu.g of DNA (.gamma.-.sup.32 P) ATP: 3000Ci/mmol, 10 .mu.Ci/.mu.l, with 6
.mu.l/40 .mu.l of reaction mixture being used). The probes had a specific
activity of 1.times.10.sup.8 Bq/.mu.l or 1.5.times.10.sup.6 Bq/pmol.
9. Screening of the Placental cDNA With PP4-Specific Oligonucleotides
1.times.10.sup.6 PFU of the placental cDNA bank were examined with the PP4
oligonucleotide probes 104 and 125 together. For this purpose,
3.times.10.sup.4 PFU were plated out with cells of the E. coli K 12 strain
C 600 HFL in soft agar on 13.5 cm petri dishes and incubated at 37.degree.
C. for 6 h. Lysis was still incomplete at this time. The plates were
incubated in a refrigerator overnight, and the phages were transferred to
nitrocellulose filters (Schleicher & Schull, BA 85, Ref.-No. 401124)
(duplicates). The nitrocellulose filters and Petri dishes were marked with
an injection needle to allow assignment later. During the processing of
the nitrocellulose filters, the Petri dishes were stored in a cold room.
The DNA on the nitrocellulose filters was denatured by placing the filters
on filter paper (Whatman M3) impregnated with 1.5M NaCl, 0.5M NaOH for 5
min. The filters were then renatured in the same way using 1.5M NaCl, 0.5
M Tris (pH 8.0) and washed with 2.times.SSPE (0.36M NaCl, 16 mM NaOH, 20
mM NaH.sub.2 PO.sub.4, 2 mM EDTA). The filters were then dried in vacuo at
80.degree. C. for 2 h. The filters were washed in 3.times.SSC, 0.1% SDS
(20.times.SSC=3M NaCl, 0.3M Na citrate) at 65.degree. C. for 4 h and
prehybridized at 65.degree. C. for 4 h (prehybridization solution: 0.6M
NaCl, 0.06M Tris (pH 8.3), 6 mM EDTA, 0.2% non-ionic synthetic sucrose
polymer (.sup.R Ficoll), 0.2% polyvinylpyrrolidone 40, 0.2% BSA, 0.1% SDS,
50 .mu.g/ml denatured herring sperm DNA). The filters were incubated
overnight with the addition of 100,000-200,000 Bq of the labeled
oligonucleotide per ml of hybridization solution (as prehybridization
solution but without herring sperm DNA) in beakers or in sealed
polyethylene films, shaking gently. The hybridization temperature was
44.degree. C. The nitrocellulose filters were washed with 6.times.SSC,
0.05M sodium pyrophosphate at room temperature for one hour and at the
relevant hybridization temperature for a further hour. The filters were
dried and autoradiographed overnight. Signals which appeared on both
duplicates of the X-ray film were assigned to the Petri dish, and the
region (about 50 plaques) was punched out with the wide end of a Pasteur
pipette, and the phages were resuspended in 1 ml of SM buffer. Positive
phages were singled out over three cycles until a single clone was
obtained.
In total, 1.times.10.sup.6 PFU of the placental cDNA bank were examined. 54
signals were identified on duplicate filters. On further screening with
the individual probes, it emerged that nine clones reacted with both
probes. All these nine clones carry a PP4-X coding sequence, with a
maximum length of 1326 base-pairs for the clone PP4-X/23. Sequence
analysis of the PP4-X clones subsequently showed that eight mismatches
with the PP4-X sequence which has been found occur over the complete
length of each of the two probes (Table 1).
TABLE 1
__________________________________________________________________________
PP4-X seq. versus PP4 oligonucleotide 104
##STR1##
PP4-X seq. versus PP4 oligonucleotide 125
##STR2##
__________________________________________________________________________
10. DNA Sequence Analysis
The phage clones PP4-X/10, /23, /47 and /49 were propagated, and the DNA of
each of them was extracted. In each case the EcoRI fragment was isolated
and ligated into the EcoRI sites of the vector pIC19H for restriction
analyses and of the vector M13mp8 for sequence analyses using the
enzymatic dideoxy method of Sanger. The sequence shows an open reading
frame and codes for a protein having a maximum of 321 amino acids (FIG.
2).
11. Expression of PP4-X in E. coli
a) Expression of the mature unfused PP4-X protein:
The expression vector pTrc97A (European Patent Application EP 0 236 978)
was digested with EcoRI and the staggered 5' ends were eliminated with the
single strand specific enzyme mung bean nuclease, thus giving the
sequence: 5' AACAGACCATGG 3'. Subsequently the DNA was digested with
HindIII and the 2 889 bp fragment was isolated. This fragment was ligated
with the 1165 bp BalI/HindIII fragment of the PP4-X cDNA in that the blunt
end produced by BalI of the cDNA reacted with the blunted EcoRI end of the
vector. The resulting sequence coding for the N-terminal amino acids of
PP4-X are:
##STR3##
The resulting 4054 bp expression plasmid pPP4-X is thus capable to allow
the sythesis of the mature unfused PP4-X protein in E. coli.
In order to express the PP4-X protein the E. coli K12 strain W31101acIQ was
transformed by pPP4-X DNA in the usual way. Ampicillin-resistant colonies
express the expected PP4-X protein after induction. Analysis of cellular
expression products after SDS-PAGE and staining of bands with Coomassie
blue showed a new band of a position corresponding to a molecular weight
of approx. 36 kD. Analysis of control cells which were not transformed
with pPP4-X DNA do not show this band. The protein which appears in the
W31101acIQ (pPP4-X)-extracts reacts in a Western Blot experiment
specifically with anti-PP4 antisera. In a further experiment the cellular
proteins of the bacterial culture were labeled with .sup.35 S-methionin
after induction by isopropylthiogalactoside (IPTG) and later analysed by
SDS-PAGE followed by autoradiography. Again a prominent protein band of
approx. 36 kD showed up. This protein is immunoprecipitated by anti-PP4-X
antisera.
b) Expression of PP4-X fusion proteins
In a further experiment a PP4-X fusion protein with E. coli
beta-galactoside was made. For that purpose plasmid p BD2IC20H (European
Patent Application (0 236 978) was digested with SmaI and HindIII, the 3.8
kb fragment was isolated and ligated with the BalI/HindIII fragment of the
PP4-X cDNA mentioned under (a). The resulting plasmid pBD2IC20H-PP4-X
expressed in E. coli a fusion protein of approx. 77 kD which consists of
the beta-galactosidase part of approx. 41 kD and of the PP4-X part of 36
kD. This fusion protein is highly expressed and reacts likewise with
anti-PP4-X antisera.
Additionally a PP4-X fusion protein with bacteriophage MS2 DNA polymerase
was made. To that end plasmid pEx31c (K. Strebel et al. (1986), J. Virol.
57, 983-991) was digested with EcoRI and the DNA was made blunt-ended with
Klenow-polymerase. Subsequently the DNA was digested with HindIII, the 3.2
kb fragment was isolated and ligated with the above-mentioned BalI/HindIII
fragment of the PP4-X cDNA. After induction by temperature shift this
protein is likewise highly expressed and reacts also specifically with
anti-PP4-X antisera.
Both PP4-X fusion proteins are suitable means to produce and to detect
antibodies to PP4-X.
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